Lambda phage display system and the process

ABSTRACT

The present invention relates to a process of obtaining recombinant lambdoid bacteriophage with high density display of functional peptides and proteins on surface of said phage comprising of: constructing a donor plasmid having a nucleotide sequence that defines the elements for replication of the vector in bacteria, a selectable marker, a nucleotide sequence flanked by two non-compatible recombination sequences, and an inducible cistron for expression of a capsid protein and a fusion protein; constructing a recipient phage having a nucleotide sequence that defines the lambdoid elements for replication and packaging of the vector into an assembled bacteriophage and encodes an inducible cistron for expression of a selectable marker flanked by two non-compatible recombination sequences; transferring the said donor plasmid to said recipient plasmid to obtain cointegrates; growing said cointegrates in selective liquid medium; harvesting phages displaying protein encoded by the foreign DNA encapsulated in said harvested phage particle.

FIELD OF INVENTON

This invention relates to a process of obtaining recombinant lambdoidbacteriophage and the resultant novel phage display system.

BACKGROUND OF INVENTION

Identification of specific binding sequences is an integral part ofresearch; delineation of antibody recognition sites, study ofhost-pathogen interaction, elucidation of signal transduction pathways,understanding of cellular responses, all these involve interaction ofvarious macromolecules with one another; most of these interactionsoccurring between proteins or of a protein with another macromolecule.

For several years, expression libraries have been used for isolatingligands of desired specificity. However, the cumbersome technique ofimmobilizing expressed proteins on filters and screening large number offilters to obtain one specific binder is labour intensive andexhaustive, limiting the library size and number of clones that can bescreened.

It was the concept of phage display introduced by Smith in 1985 (Smith,1985), which revolutionized the field of proteomics. Large librariescame to be made in phage, and specific binders could be enriched from amilieu of millions under user-defined conditions. DNA encoding theselected molecule present inside the displaying phage particle couldthen be sequenced to know the identity of the binder. It was this beautyof the phage display system that led to an upsurge in the use of thistechnology in almost every field of science and unraveling of a plethoraof applications, which are increasing everyday. Not limited to phagetoday, display of peptides/proteins on surface of bacteria, yeast andeukaryotic cells has also come into use.

The filamentous bacteriophage M13, with which was introduced the conceptof display system is to date the most widely used system for display ofpeptides/proteins. Fusion to the minor coat protein, gIIIp and majorcoat protein, gVIIIp have beep used for display of a range of moleculesof different sizes and structure (McCafferty et al., 1990; Scott andSmith, 1990; Kang et al., 1991). Simple biology, ease of culturing andisolating phages, small genome allowing easy manipulations, welldocumented protocols have all contributed to the immense popularity ofthe M13 system.

However, M13 morphogenesis occurs in the periplasm, therefore, it isessential that the molecules to be displayed be secretion competent.Though, versions of M13 which allow C-terminal display (Crameri andBlaser, 1996), have been developed, M13 continues to be used primarilyas an N-terminal display system. Therefore, it is not useful forstudying interactions involving free-C-terminus of display partner andfor making full-length cDNA libraries from polyA mRNA.

One system, which obviates these problems associated with M13 display,is lambda display system. Phage lambda assembles in host cytosol and canbe used for both N- and C-terminal display of molecules. There have beenfew papers in recent years that have described display ofpeptides/proteins on phage lambda as fusion to the capsid protein ‘d’and tail protein ‘v’ (Dunn, 1995; Sternberg and Hoess, 1995; Mikawa etal., 1996). However, this system has not gained much popularity, whichcan be attributed to several reasons including the fact that

-   (i) the lambda phage biology is more complex than of M13 phage.    Unlike M13 which grows by extruding from host cell, lambda can    follow a lysogenic or lytic mode; therefore manipulation of lambda    life cycle is more difficult,-   (ii) lambda genome is very large (50 kb), therefore isolation of    viral DNA, insertion of user defined restriction sites, cloning of    foreign fragments and then packaging of the ligated product in vitro    to make lambda particles is difficult and the library sizes achieved    are less than those obtained with phage/phagemid based M13 vectors.

SUMMARY OF THE INVENTION

Accordingly the instant invention describes a phage lambda displaysystem which allows high efficiency cloning in phage lambda usinglox-cre recombination system and high density display ofprotein/peptides fused to the head protein, ‘d’, of phage lambda.Further, the display of different molecules on phage lambda is comparedwith display of same molecules on phage M13.

The instant invention provides for a novel system for C-terminal displayon phage lambda that avoids cloning into lambda phage DNA. ThisC-terminal display system achieves cloning efficiencies comparable tothose obtained with any plasmid system and eliminates the step of invitro packaging of phage lambda. The high-density display of foreignproteins is useful in studying low affinity protein-proteininteractions, which is more efficient as compared to M13 display system.

The object of the instant invention is to provide a system for obtainingrecombinant lambdoid bacteriophage with high-density display offunctional peptides and proteins that has high efficiency cloning inphage lambda.

Another object is to provide a method for detecting the presence ofinsert-encoded polypeptide in a given sample.

Yet another object is to create a library of cointegrates and maintainthe same library size in the lambda genome as obtained during cloning inthe donor plasmid.

Another object of the instant invention is to provide a method foridentification of the insert-encoded polypeptide by sequencing theinsert DNA in the cointegrate.

Accordingly the instant invention provides for a process of obtainingrecombinant lambdoid bacteriophage with high density display offunctional peptides and proteins on surface of said phage comprising of:

-   -   constructing a donor plasmid having a nucleotide sequence that        defines the elements for replication of the vector in bacteria,        a selectable marker, a nucleotide sequence flanked by two        non-compatible recombination sequences, and an inducible cistron        for expression of a capsid protein and a fusion protein.    -   constructing a recipient phage having a nucleotide sequence that        defines the lambdoid elements for replication and packaging of        the vector into an assembled bacteriophage and encodes an        inducible cistron for expression of a selectable marker flanked        by two non-compatible recombination sequences,    -   transferring the said donor plasmid to said recipient plasmid to        obtain cointegrates    -   growing said cointegrates in selective liquid medium    -   harvesting phages displaying protein encoded by the foreign DNA        encapsulated in said harvested phage. particle.

The instant invention also provides for a library of cointegratecontaining recombinant phage particles wherein each particle contains arecombinant lambdoid bacteriophage vector comprising the donor plasmidvector integrated into recipient phage genome.

Further the instant invention also provides for a method for detectingthe presence of insert-encoded polypeptide in a sample.

The instant invention also provides for a method for producing morecopies of the selected recombinant phage particles.

The said invention provides for a method for identification of theinsert-coded polypeptide by sequencing the insert DNA in the saidcointegrate.

A novel donor plasmid.

A novel recipient phage

A novel DCO (double crossover) cointegrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) shows a diagrammatic representation of donor plasmid,pVCDcDL1.

FIG. 1(B) shows the diagrammatic representation of recipientbacteriophage Lambda, λDL1.

FIG. 1(C) shows sequences of lox P_(wt) and lox P₅₁₁ sites are shown.The difference between lox P_(wt) and lox P₅₁₁ is shown in bold letters.

FIG. 2 shows the diagrammatic representation of recipient bacteriophageLambda, λDL1

FIG. 3 shows ELISA reactivity of lambda phages displaying cmyc peptide

FIG. 4 shows the diagrammatic representation of various peptides fusedto coat proteins of bacteriophage M13 and λ.

FIG. 5 shows the comparison of ELISA reactivity of various phagesdisplaying p24 and its fragments

FIG. 6 shows the Western blot analysis of M13 and lambda phagesdisplaying HIV-1 p24 and its fragments.

FIG. 7 shows the comparison of display density on Lambda and M13 phagesFIG. 8A shows the alignment of inserts in the selected phage FIG. 8Bshows the reactivity of phages in ELISA FIG. 9 shows the binding oflambda phages displaying a single chain Fv (scFv)

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1(A) shows the diagrammatic representation of donor plasmidpVCDcDL1. The diagram shows only relevant genes. The backbone is derivedfrom pUC119 and shows Lac PO, Lac Promoter—operator; Amp^(r), βlactamase gene, F ori, original of replication derived from filamentousbacteriophage; LoxP_(wt), wild type Lox P site, LoxP₅₁₁, mutant Lox Psite, D, DNA encoding gpD of bacteriophage Lambda, collagenase, sequencecleaved by enzyme collagenase, MCS (multiple cloning site); tag, a 10amino acid sequence from cmyc that is recognised by Mab, 9E10.

FIG. 1(B) shows the diagrammatic representation of recipientbacteriophage Lambda, λDL1. The diagram shows a DNA cassette containingcomplete transcription and translation unit of lacZα flanked byLoxP_(wt) (Wild type LoxP). LoxP₅₁₁, (mutant LoxP) has been inserted inthe unique Xba I site; D_(am) is the D gene of Lambda with ambermutation.

Fig 1(C) shows sequences of LoxP_(wt) and LoxP₅₁₁ site. The differencebetween LoxP_(wt) and LoxP₅₁₁ is shown in bold letters. The sequencesshown in FIG. 1 (C) are SEQ ID NO:1 (ATAACTTCGT ATAATGTATG CTATACGAAGTTAT), SEQ ID NO:2 (ATAACTTCGT ATAGCATACA TTATACGAAG TTAT). SEQ ID NO:3(ATAACTTCGT ATAATGTATA CTATACGAAG TTAT). and SEQ ID NO:4 (ATAACTTCGTATAGTATACA TTATACGAAG TTAT).

FIG. 2 shows the process of recombination. The donor plasmid e.g.pVDcDL1 is transformed in Cre expressing E. coli strain, BM 25.8. Theculture of transformants is infected with recipient bacteriophage λDL1,which does not display any protein fused to gpD. Single cross over (SCO)or double cross over (DCO) event takes places and integration of plasmidconfers Ampicillin resistance. The Amp^(r) clones are grown to producebacteriophage lambda displaying desired protein fused to gpD.Description of various gene segments is given in FIG. 1.

FIG. 3 shows the binding of λDcDL1 phages to Mab 9E10. The microtitreELISA plates were coated with Mab 9E10 (100 microlitre/well of 1:1000dilution of ascitic fluid). Indicated number of purified phages wereadded and incubated for 1 hr at 37° C. The bound phages were detected byadding Rabbit anti-λ polyclonal serum followed by HRP-conjugated Goatanti-Rabbit IgG (H+L).

FIG. 4 shows the diagrammatic representation of various peptides fusedto coat proteins of bacteriophage M13 and Lambda. These peptides arep24, HIV-1 capsid protein p24 of 231 amino acids; p246, amino acids 1 to156 of HIV-1 p24; p241, amino acids 1 to 72 of HIV-1 p24. D, protein gpDof bacteriophage lambda; g8p, gene 8 protein of phage M13; g3p, gene 3protein of phage M13; cmyc, a 10 amino acid peptide tag recognized byMAb 9E10. The tabulated data also shows the molecular weights of fusionproteins displayed on phage surface along with length of displayed p24sequence.

FIG. 5 is a comparative chart depicting the comparison of ELISAreactivity of various phages displaying p24 and its fragments.Microtitre ELISA plates were coated with MAb H23 (1:1000 dilution ofascitic fluid). Different dilutions of phages were incubated for 1 hr at37° C. The bound phages were detected by addition of Rabbit anti-M13 orRabbit anti-Lambda polyclonal sera followed by HRP conjugated Goatanti-Rabbit IgG (H+L). p24-g8p, M13 phages displaying fusion proteinbetween 1-72 amino acids of HIV-1 p24 fused at the N-terminus of g8p;p241-g3p, same as p241-g8p but with fusion at the N-terminus of g3p;p246-g8p, M13 phages displaying protein between 1-156 amino acids ofHIV-1 p24 fused at the N-terminus of g8p; p246-g3p, same as p246-g8p butwith fusion at the N-terminus of g3p; p24-g8p, M13 phage displayingHIV-1 p24 (amino acids 1-231) fused at the N-terminus of g8p; p24-g3p,same as p24-g8p but with fusion at the N-terminus of g3p; D-p241, lambdaphages displaying fusion protein of 1-72 amino acids of HIV-1 p24 at theC-terminus of gpD; D-p246; same as D-p241 but containing 1-156 aminoacids of HIV-1 p24; D-p24, same as D-p241 but containing full length p24(amino acids 1-231).

FIG. 6 shows the results of the Western blot analysis of M13 and lambdaphages displaying HIV-1 p24 and its fragments. Purified phages weresubjected to SDS-PAGE at indicated acrylamide concentration underreducing conditions. After transferring on to PVDF membrane, the blotswere probed with anti-cmyc MAb 9E10 followed by HRP-conjugated Goatanti-Mouse IgG (H+L).

-   A. 1×10⁸ gpD displaying lambda phages on 12.5% PAG;-   B. 2×10¹¹ g8p displaying M13 phages per lane on 12.5% PAG;-   C. 1×10¹¹ g3p displaying M13 phages per lane on 10% PAG; Lane 1 is    for lambda phage displaying D-cmyc fusion protein; Lane 2, 5, and 8,    are for phages displaying fusion protein of 1-72 amino acid of    HIV-p24 (p241) fused to respective phage coat protein; lane 3, 6,    and 9 are for phages displaying fusion protein of 1-156 amino acids    of HIV-1 p24 (p246) fused to phage coat proteins and lane 4, 7, and    10 are for phages displaying fusion proteins of HIV-1 p24 (231 amino    acids) fused with various coat proteins.

FIG. 7 shows the tabulated data comparing the display density on Lambdaand M13 phages. Western blots as shown in FIG. 6 were subjected todensitometric scanning. Calculation of number of displayed fusionproteins per phage particle was made by estimating the number ofmolecules in the band corresponding to the fusion protein in comparisonto known amount of purified GST-cmyc analysed on the same blot.

FIG. 8. A shows the alignment of inserts in phage selected after panningof PE gene-fragment library in M13 and λ phage on human serum. Only 15clones (out of 30 clones) of lambda are shown. Solid line representsPE-38 and the numbers above the line denote amino acid position in thefull length PE. The clones selected for ELISA (Panel B) are indicated as‘a’ to ‘d’.

FIG. 8B ELISA showing reactivity of phages displaying PE fragments.Reactivity of M13 phage, clone a and b and lambda phage, clone c and dto pre-treatment (open symbols) and post-treatment (filled symbols)samples.

FIG. 9 depicts the binding of Lambda phages displaying a single chain Fv(scFv). Microtitre ELISA plates were coated with mesothelin (100microgram/well). Indicated number of purified λDcSS1DL1 phagesdisplaying SS1scFv, λDcDL1 (control phage) displaying cmyc peptide andλDL1 (control phage) displaying no peptide were added. The bound phageswere detected by adding Rabbit anti—λ polyclonal serum followed byHRP-conjugated Goat anti-Rabbit IgG (H+L).

DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO THE AccompanyingDrawings

The described system for display on phage lambda utilizeshigh-efficiency plasmid based cloning protocols followed by in vivorecombination process for cloning of desired DNA in lambda genome. Inthis system, the DNA encoding molecule to be displayed is cloned in adonor plasmid using conventional cloning protocols. Cre expressingbacteria are then transformed with this donor plasmid. When thetransformed cells are infected with recipient lambda particles, thedonor plasmid undergoes Cre mediated recombination with lambda DNA atthe Lox sites and gets integrated into the lambda genome.

This λ cointegrate formed by recombination expresses d-fusion protein(FIG. 2). When lambda morphogenesis occurs and phage assembly takesplace, the λ cointegrate DNA gets packaged in pre-formed head and theD-fusion protein gets incorporated in the newly formed phage capsid. Theprogeny particles therefore give ampicillin resistant transductants(allowing easy selection) and display D-fusion protein.

The use of in vivo recombination process for transferring DNA fromplasmid into the lambda genome maintains the library size obtainedduring cloning in donor plasmid.

The invention shall now be described with the help of some examples:

EXAMPLE 1

The system described above is optimized using donor plasmid pVCDcDL1,which expresses D protein fused to a ten amino acid tag, cmyc. pVCDcDL1was used to transform BM25.8 (Cre+) cells and the transformants wereinfected with recipient lambda, λDL1. The lysate obtained after culturegrowth and lysis was plated to obtain ampicillin resistanttransductants. These transductants were analysed by PCR to check forintegration of plasmid into λ DNA.

Two kinds of cointegrates were obtained. Single crossover at one of thelox sites resulted in formation of SCO (single crossover cointegrates)which contained the entire donor plasmid and lacZα cassette (FIG. 2). Asecond crossover event resulted in excision off ori and lacZα cassetteand this was called DCO (double crossover cointegrates). Both SCO andDCO produced same number of phages and the display of D-cmyc fusionprotein was comparable.

DcDL1 phages were made and tested for display of D-cmyc in ELISA.Purified DcDL1 phages were added to microtitre wells coated withanti-cmyc antibody, 9E10. DL1 phages were used as Control.

DcDL1 phages showed dose-dependent binding to 9E10, (as seen in FIG. 3)showing that they displayed D-cmyc fusion protein. No binding wasobserved with DL1 phages that do not display D-cmyc fusion protein.

To study the display of different size molecules as d-fusion protein onlambda, we cloned different length fragments of HIV-1 capsid protein,p24 in pVCDcDL1. The recombinants were named pVCDc(p241)DL1,pVCDc(p246)DL1 and pVCDc(p24)DL1. The three constructs were then used totransform host BM25.8 and recombination was carried out to obtainDc(p241)DL1, Dc(p246)DL1 and Dc(p24)DL1.

These phages were purified and characterized for reactivity to anti-p24antibody in ELISA and for display density by Western blot. Also, thesame fragments of p24 were also cloned in M13 g3p and g8p displayvectors for a comparative study.

FIG. 4 shows the various fragments displayed along with their molecularweight. In ELISA, p24 displaying and M13 phages were captured on wellscoated with anti-p24 Mab, H23 and then bound phages were detected usinganti-phage polyclonal sera. For all the three fragments displayed,phages showed better reactivity, 2-3 orders of magnitude more than thecorresponding M13 (g8p and g3p display) phages (FIG. 5). To quantitatethe number of displayed molecules per phage particle, Western blotanalysis was done. Known number of purified phages were electrophoresedon SDS-PAG and transferred to PVDF membrane and probed with anti-cmycMab, 9E10 (FIG. 6). Purified GSTcmyc (Glutathione S-transferase fused tocmyc) was used as control to quantitate the bands obtained. The resultsare shown in FIG. 7. Lambda phages were found to display several foldsmore amount of protein than M13 phages. As seen in FIG. 7, lambda wasfound to display 100 times more copies of p241 compared to g⁸p and g3pdisplay phages. The difference was greater with larger size fragments,p246 and p24.

These results clearly show that for all fragment sizes, the displaydensity on lambda is 2-3 orders of magnitude more than on M13. Thissystem will be useful in studying weak interactions and in epitopemapping where high-density display is advantageous.

M13 assembly occurs in the periplasm of the cell. The oxidisingenvironment of the periplasm allows formation of disulfide binds in thedisplayed molecules. Lambda assembles in the cytosol and therefore,disulfide bonded molecules may not be displayed in functional form onlambda.

To check this, we displayed single chain fragment (scFv) of ananti-mesothelin antibody, SS1 as D-fusion protein on lambda. DcSS1DL1was checked for functional display of scFv in ELISA. DcSS1DL1 phageswere added to mesothelin coated wells and captured phages were detectedusing anti-phage polyclonal sera. As shown in FIG. 8, DcSS1DL1 phagesshowed specific binding to mesothelin, indicating that the displayedscFv was functional. No binding was observed with DL1 and DcDL1 phages.This shows that disulfide containing peptides and proteins can bedisplayed in functional form on lambda phage.

EXAMPLE 2

The ‘Donor’ plasmid vector pVCDcDL1, (FIG. 1A) a high copy number vectorbased on pUC119, was constructed for cloning sequences to be displayedas d-fusion protein on. This plasmid vector contains ‘d’ gene of underlacPO followed by a Gly-Ser spacer, collagenase cleavage site, multiplecloning site (MCS) and a decapeptide tag, cmyc. Cloning of DNA sequencesin the MCS allows formation of d-fusion protein with the fusion proteinpresent at C-terminus of d protein. The collagenase site allows cleavageof the fusion partner from d protein and is used for elution of boundphages after panning by collagenase treatment. The cmyc tag allows easyidentification of the fusion protein. The vector contains M13 phageorigin of replication (f ori), flanked by Lox P_(wt) and Lox P₅₁₁ sites(Hoess et al., 1982).

This cassette is extremely stable and there is no loss of ‘f ori’sequence upon repeated cycles of growth (more than five times) inCre⁺/Cre⁻host (data not shown).

pVCDcDL1 was analysed for the expression of d-cmyc protein. It was foundthat ˜50% of total cellular protein in TG1 cells on IPTG induction wasd-cmyc fusion protein. Localization experiments showed that all theexpressed protein was present in soluble fraction of cell cytoplasm(data not shown).

For preparing the recipient ‘lambda’ vector a sequence coding for lacZand flanked by lox sites (FIG. 1C), was cloned in unique Xba I site ofDam (Stemberg and Hoess, 1995). This vector was named DL1 (FIG. 1B).This vector has ‘d’ gene carrying amber (TAG) codon.

In vivo recombination of DL-1 and pVCDcDL1 produced cointegratesdisplaying fusion protein.

BM 25.8 (Cre⁺) cells were transformed with plasmid pVCDcDL1 and grown toO.D. ˜0.3 in LB Amp₁₀₀ (LB medium containing 100 g/ml of ampicillin).The cells from 1 ml culture were harvested and resuspended in 100 IofDL-1 phage at an MOI of 1.0. After incubation at 37° C. for 10 minutes,the sample was diluted in 1 ml LB Amp₁₀₀ MgCl₂ (10 mM), IPTG (1 mM) andgrown at 37° C. with shaking till lysis occured (˜3-4 hours). Thecell-free supernatant was titrated on TG1 (Cre⁻) cells and Amp^(r)colonies obtained were analysed by PCR using specific primers L1 and L4to identify lambda cointegrates, DcDL1.

Amp^(r) colonies of cointegrate were grown in LB Amp₁₀₀ and thesupernatant containing phages was titrated to obtain plaques. 0.1 ml TG1cells were infected with a single plaque and then diluted in 5 ml LB andthe culture was grown at 37° C. for 3-4 hours or till lysis wasobserved. CHCl₃ was then added (final concentration 1%) to completelysis and the cell-free supernatant was titrated to determine phagenumbers.

For large-scale purification of phages, 15 ml TG1 cells were infectedwith phages at MOI of 0.01, diluted to one litre in LB medium and grownat 37° C. till lysis occured. The cell-free supernatant was then treatedwith DNase/RNase. PEG-NaCl was added to a final concentration of 10%PEG/1M NaCl and kept a 4° C. for 4 hrs. The phages were harvested bycentrifugation at 20,000×g for 30 minutes and then suspended in 10 mMTris containing MgCl2 and 0.1% gelatin (TMG). The phage suspension wasthen subjected to ultra centrifugation at 50,000 rpm (70 Ti) for 3hours. The phage pellet was suspended in TMG and re-centrifuged at50,000 rpm. The pellet was then suspended in TMG and phage suspensionwas titrated for pfu (plaque forming units).

EXAMPLE 3

Comparison of Lambda Display System with M13 Display System

DNA sequences encoding different length fragments of HIV capsid proteinp24 were amplified from pVCp24210 (Gupta et al., 2000) using 5′ primerwhich annealed to first eight codons of the fragment and contained Nhe Isite and 3′ primer which annealed to last eight codons of the fragmentand contained Mlu I site. The amplified products were digested with NheI and Mlu I and cloned in Nhe I-Mlu I digested, dephosphorylated vector,pVCDcDL1 to create pVCDc(p241)DL1, pVCDc(p246)DL1 and pVCDc(p24)DL1.

The three constructs were then used to transform Cre⁺ host BM25.8 andrecombination was carried out to obtain Dc(p241)DL1, Dc(p246)DL1 andDc(p24)DL1.

DNA encoding different p24 fragments were cloned as Nhe I-Mlu I insertsinto gIII display vector, pVC3TA726 (Sampath et al., 1997) and similargVIII display vector, pVCp2418426 (VKC, personal communication). Therecombinants pVC(p241)3426, pVC(p246)3426 and pVC(p24)3426 (displayingp241, p246 and p24 as gIIIp fusion) and pVC(p241)18426, pVC(p246)18426and pVC(p24)18426 (displaying p241, p246 and p24 as gVIIIp fusion) weregrown and phage particles were made after infection with helper phage,VCSM13. The phages were precipitated with PEG-NaCl and purified byultracentrifugation. The purified phages were then titrated as Amp^(r)transductants cfu (colony forming units).

Ultra-purified phages were used for western blot analysis. Phages weremixed with 2× Lamelli buffer containing ME and electrophoresed on 10%SDS-12.5% PAG/10% PAG. The samples were then transferred onto PVDFmembrane (immobilon, Millipore) and probed with 1:1000 dilution ofanti-cmyc MAb, 9E10/anti-p24 MAb, H23 followed by 1:2500 dilution of HRPconjugated Goat anti-Mouse IgG (H+L).

For ELISA, microtitre wells were coated with 1:1000 dilution of MAb 9E10or H23 and ultra-purified phages were added to the coated wells. Thebound phages were detected with Rabbit anti-polyclonal serum or Rabbitanti-M13 polyclonal serum followed by HRP conjugated Goat anti-RabbitIgG (H+L). Construction of Pseudomonas Exotoxin (PE) Gene-fragmentLibrary in λ and M13 Vectors. Fifty-200 bp random fragments of DNAencoding PE-38, a 38 KDa fragment of P E Debinski and Pastan, 1992 wereproduced by Dnase I digestion and ligated as blunt-ended fragments inpVCDcDL3 using previously described protocols Gupta et al., 2001. Theligation mix was electroporated into BM25.8 cells and plated on two 150mm LBAmpGlu (LBAmp medium containing 1% Glucose) plates on to obtain5×10⁶ independent clones. The transformants were scrapped and suspensionstored at −70° C. Cells (1×10⁹) of the library were grown in 10 mlLBAmpGlu to an OD₆₀₀ of 0.3, harvested and suspended in 1 ml of λDL1phage lysate at an MOI of 1.0. After incubation at 37° C. for 10 min,the samples were diluted in 10 ml LBAmp containing MgCl2 (10 mM) andgrown at 37° C. with shaking for 3 h until cell lysis. The cell-freesupernatant pfu (10 ml) was used to infect an exponential phase cultureof TG1 cells (10 ml) and plated on twenty 150 mm LBAmpGlu plates. TheAmp^(r) colonies obtained were scraped and stored at −70° C. Cells(1×10⁹) harbouring cointegrates were diluted into 50 ml of LBAmp mediumand grown at 37° C. for 8 h to produce phage. The cell-free supernatantwas directly used for affinity selection.

PE-derived fragments were also ligated to Sma I digested gIIIp basedphagemid display vector, pVCEPI13426 Gupta et al., 1999 to obtaingene-fragment library in M13. A library size of 6×10⁶ independent cloneswas obtained in TG1 cells and was used to produce phage as describedpreviously Kushwaha et al., 1994.

EXAMPLE 4

Affinity Selection of Binders by Bio-Panning.

For bio-panning, wells of microtiter plates were coated with 1:1000dilution of ascitic fluid of anti-cmyc MAb 9E10 and phage lysate wasadded to the coated wells Gupta et al., 1999. The captured λ phages wereeluted with Collagenase. 1 unit of Collagenase was added to each welland incubated for 30 min at RT. The eluted phages were serially dilutedand used to infect TG1 cells and plated to determine phage infectedcells as pfu and cfu. For M13 library, the captured phage were elutedusing low pH buffer Gupta et al., 1999 and titrated on TG1 as cfu. Forpanning of PE gene-fragment library on MAb, wells were first coated withgoat anti-mouse IgG followed by 1:100 dilution of anti-PE MAb culturesupernatant (Test wells) or buffer (Control wells). For panning of thislibrary on human serum, wells were coated with Goat anti-human (IgG+IgM)followed by 1:100 dilution of serum from patients treated with PE-basedimmunotoxins (Test wells) or pre-treatment serum of patients (Controlwells). Phage lysate was added to each well, incubated at 37° C. for hrand unbound phages were removed by washing as described previously (b).Individual Amp^(r) colonies were grown and phage particles were producedas described previously Kushwaha et al., 1994. The cell-freesupernatants were used for ELISA.

Cell-free supernatants of individual phage clones selected inbio-panning experiments were tested in ELISA. For this, wells werecoated with 1:1000 dilution of rabbit anti-λ polyclonal serum or rabbitanti-M13 polyclonal serum and phage were added to the coated wells.After removing unbound phage, 1:100 dilution of anti-PE MAb (culturesupernatant) or serum from patients treated with PE-based immunotoxinswas added. The bound phage were detected with HRP conjugated goatanti-mouse IgG (H+L)/HRP conjugated goat anti-human (IgG+IgM).

EXAMPLE 5

Display of Antibody Fragments on Phage Lambda

DNA encoding scFv fragment of anti-mesothelin antibody, SS1 (Chowdhuryand Pastan, 1999) was amplified from pVNLSS11346 and cloned as Nhe I-MluI insert in pVCDcDL1, to obtain pVCDcSS1DL1. BM25.8 cells weretransformed with pVCDcSS1DL1 and recombination was performed using DL1as described above to form DcSS1DL1. These phages were purified usingprotocol described above for DcDL1.

ELISA was performed to check functionality of displayed scFv. For this,microtitre wells were coated with 100 ng of mesothelin andultra-purified phages were added to the coated wells. The bound phageswere detected with Rabbit anti polyclonal serum followed by HRPconjugated Goat anti-Rabbit IgG (H+L).

The described system for display on phage lambda utiliseshigh-efficiency plasmid based cloning protocols followed by in vivorecombination process for cloning of desired DNA in lambda genome. Inthis system, the DNA encoding molecule to be displayed is cloned in adonor plasmid using conventional cloning protocols. Cre expressingbacteria are then transformed with this donor plasmid. When thetransformed cells are infected with recipient lambda particles, thedonor plasmid undergoes Cre mediated recombination with lambda DNA atthe Lox sites and gets integrated into the lambda genome. Thiscointegrate formed by recombination expresses d-fusion protein (FIG. 2).When lambda morphogenesis occurs and phage assembly takes place, thecointegrate DNA gets packaged in pre-formed head and the D-fusionprotein gets incorporated in the newly formed phage capsid. The progenyparticles therefore give ampicillin resistant transductants (allowingeasy selection) and display D-fusion protein. The use of in vivorecombination process for transferring DNA from plasmid into the lambdagenome maintains the library size obtained during cloning in donorplasmid.

This system was optimised by using donor plasmid pVCDcDL1, whichexpresses D protein fused to a ten amino acid tag, cmyc. pVCDcDL1 wasused to transform BM25.8 (Cre+) cells and the transformants wereinfected with recipient lambda, DL1. The lysate obtained after culturegrowth and lysis was plated to obtain ampicillin resistanttransductants. These transductants were analysed by PCR to check forintegration of plasmid into DNA. Two kinds of cointegrates wereobtained. Single crossover at one of the lox sites resulted in formationof SCO (Single crossover cointegrates) which contained the entire donorplasmid and lacZ cassette (FIG. 2). A second crossover event resulted inexcision of f ori and lacZ cassette and this was called DCO (doublecrossover cointegrates). Both SCO and DCO produced same number of phagesand the display of D-cmyc fusion protein was comparable. DcDL1 phageswere made and tested for display of D-cmyc in ELISA. Purified DcDL1phages were added to microtitre wells coated with anti-cmyc antibody,9E10. DL1 phages were used as Control. As shown in FIG. 3, DcDL1 phagesshowed dose-dependent binding to 9E10, showing that they displayedD-cmyc fusion protein. No binding was observed with DL1 phages that donot display D-cmyc fusion protein.

To study the display of different size molecules as d-fusion protein onlambda, different length fragments of HIV-1 capsid protein, p24 werecloned in pVCDcDL1. The recombinants were named pVCDc(p241)DL1,pVCDc(p246)DL1 and pVCDc(p24)DL1. The three constructs were then used totransform host BM25.8 and recombination was carried out to obtainDc(p241)DL1, Dc(p246)DL1 and Dc(p24)DL1. These phages were purified andcharacterised for reactivity to anti-p24 antibody in ELISA and fordisplay density by Western blot. Also, the same fragments of p24 werealso cloned in M13 g3p and g8p display vectors for a comparative study.FIG. 4 shows the various fragments displayed along with their molecularweight. In ELISA, p24 displaying and M13 phages were captured on wellscoated with anti-p24 Mab, H23 and then bound phages were detected usinganti-phage polyclonal sera. For all the three fragments displayed,phages showed better reactivity, 2-3 orders of magnitude more than thecorressponding M13 (g8p and g3p display) phages (FIG. 5). To quantitatethe number of displayed molecules per phage particle, Western blotanalysis was done. Known number of purified phages were electrophoresedon SDS-PAG and transferred to PVDF membrane and probed with anti-cmycMab, 9E10 (FIG. 6). Purified GSTcmyc (Glutathione S-transferase fused tocmyc) was used as control to quantitate the bands obtained. The resultsare shown in FIG. 7. Lambda phages were found to display several foldsmore amount of protein than M13 phages. As seen in FIG. 7, lambda wasfound to display 100 times more copies of p241 compared to g8p and g3pdisplay phages. The difference was greater with larger size fragments,p246 and p24.

These results clearly show that for all fragment sizes, the displaydensity on lambda is 2-3 orders of magnitude more than on M13. Thissystem will be useful in studying weak interactions and in epitopemapping where high-density display is advantageous.

EXAMPLE 6

High Density Display Leads to Efficient Selection in Bio-panning.

The utility of high density display on λ in epitope mapping wasevaluated using gene-fragment library of PE carrying fragments rangingin size from 50-200 bp and encoding peptides as fusion to gpD of λ orthe gIIIp of M13. These libraries were first used to map the epitoperecognised by a MAb against PE in bio-panning. The binding of lambdalibrary phages was 2000 times more than the binding to uncoated wells,while with M13 library this ratio was only 10 times indicating highspecific binding of lambda phages (Table 1). Sequence analysis ofindividual phage from MAb-coated wells revealed that 88% of the analysedλ clones displayed a fragment of PE on their surface, as compared to 63%M13 clones.

This difference could be either due to more selective and specificbinding of λ phage or due to the fact that C-terminal display in λallows three times more functional display as compared to the N-terminaldisplay in M13 system. Lambda clones displaying a PE fragment producedhigh dose-dependent reactivity in ELISA to anti-PE MAb, while none ofthe M13 clones showed any significant reactivity despite the addition of1000-fold more M13 phage (data not shown). These results clearly showthat the number of peptides displayed on λ phage was several orders ofmagnitude higher than on M13 phage, corroborating our earlier resultsobtained with p24 displaying phage.

Anti-toxin antibodies are one of the major bottle-neck in the way ofsuccessful use of immunotoxins for cancer therapy as repeated treatmentwith immunotoxin is not possible. Therefore, identification ofimmunodominant epitopes followed by mutagenesis, might produce toxinmolecules which would exhibit full bio efficacy, but will not elicitneutralizing antibodies.

Therefore, the library was used to identify immunodominant regions of PEagainst which antibodies are present in the serum of human patientsafter treatment with recombinant immunotoxins containing PE-38 Pai etal., 1991. Microtiter wells were coated with pre-treatment (control) andpost-treatment (test) pooled sera from patients, and panning was done.

As shown in Table 2, 90% of the selected λ clones in test sample were inframe with gpD and displayed a fragment of PE, while in the case of M13phage only 16% clones were in frame with gIIlp suggesting specificenrichment in case of lambda phage. Alignment of sequence showed thatthe majority of the λ clones aligned to the last 50 amino acids of PEwhereas in the case of M13, only a few clones aligned to this region(FIG. 3A). A second round of panning with the amplified elute of firstpanning of M13 phage did not result in any increase in the number ofclones with alignment to any one region of PE (data not shown). Thisdifference between λ clones and M13 clones could be due to low ligandconcentration per phage particle in case of M13. In ELISA, the lambdaclones showed high reactivity with post-treatment serum pool and gavelow reactivity with pre-treatment serum pool (FIG. 3B). M13 phage showedreactivity with pre-treatment serum pool with only marginal increase inreactivity with post-treatment serum pool. The difference in ELISAreactivity between λ phage and M13 phage was 2-3 orders of magnitude,again establishing the importance of high density of display on λ phage.

M13 assembly occurs in the periplasm of the cell. The oxidisingenvironment of the periplasm allows formation of disulfide binds in thedisplayed molecules. Lambda assembles in the cytosol and therefore,disulfide bonded molecules may not be displayed in functional form onlambda. To check this, we displayed single chain fragment (scFv) of ananti-mesothelin antibody, SS1 as D-fusion protein on lambda. DcSS1DL1was checked for functional display of scFv in ELISA. DcSS1DL1 phageswere added to mesothelin coated wells and captured phages were detectedusing anti-phage polyclonal sera. As shown in FIG. 9, DcSS1DL1 phagesshowed specific binding to mesothelin, indicating that the displayedscFv was functional. No binding was observed with DL1 and DcDL1 phages.This shows that disulfide containing peptides and proteins can bedisplayed in functional form on lambda phage.

TABLE 1 Panning of λ and M13 Library on Anti-PE MAb λ library M13library Test Control Test Control Input phages 1 × 10⁸ 1 × 10⁸ 1 × 10¹⁰1 × 10¹⁰ Output phages 1 × 10⁴ 5 9 × 10⁵  9 × 10⁴  Fold enrichment^(*) 2× 10³ 10 Clones displaying  88% (22/25) 63% (23/36) PE fragment % (No.positive/No. analyzed)^(†) Clones ELISA reac- 100% (12/12)  0% (0/14)tive % (No. posi- tive/No. analyzed)⁵⁵⁵^(*Fold enrichment = output phage in test/output phage in Control.)^(†The inserts in clones from test well selected after panning were sequenced to check for reading frame and display of PE fragment.)^(555 The clones displaying PE fragments were analysed for binding to anti-PE MAb in ELISA.)

TABLE 2 Panning of λ and M13 Library on Human Serum λ library M13library Control Test Control Test Input phage 1 × 10⁸ 1 × 10⁸ 1 × 10¹⁰ 1× 10¹⁰ Output phage 1 × 10³ 2 × 10⁴ 6 × 10⁴  7 × 10⁶  Foldenrichment^(*) 20 123 Clones displaying PE 25% 90% 9% 15.7% fragment %(No. (8/30) (30/33) (3/34) (11/70) positive/No. analyzed)^(†) Controlrefers to pre-treatment serum and test refers to post-treatment serum ofpatients ^(* and) ^(† as in Table 1)

While particular embodiments of the present invention have beendescribed above, the present invention also pertains to novel donorplasmid as mentioned in any of the claims filed herewith. In addition,the present invention further pertains to novel recipient phage asmentioned in any of the claims filed herewith. Also, the presentinvention yet further pertains to novel DCO cointeg rate as mentioned inany of the claims filed herein.

REFERENCES

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1. A process of obtaining recombinant lambdoid bacteriophage with highdensity display of functional peptides and proteins on surface of saidbacteriophage comprising the steps of: a) Constructing a donor plasmidcomprising: A first recombination sequence, and a second recombinationsequence, wherein said first and second recombination sequences areincompatible with each other, and wherein said first and secondrecombination sequences flank a nucleotide sequence that defines theelements for replication of the vector in bacteria, a nucleotidesequence encoding a first selectable marker, and a first induciblecistron comprising: a promoter for transcribing said first cistron, afirst ribosome binding site, a first translatable nucleotide sequenceencoding a lambdoid bacteriophage capsid polypeptide a secondtranslatable sequence operatively linked to said first translatablesequence and encoding a first linker polypeptide, a protease cleavablepeptide sequence, a second linker polypeptide, and a sequence adaptedfor ligation of an insert polynucleotide or an MCS; wherein eachtranslatable nucleotide sequence is in the same reading frame such thatinduction of said first cistron expresses a fusion polypeptideconsisting of said capsid polypeptide, linker polypeptide, proteasecleavage site, linker polypeptide, and said MCS-encoded or saidinsert-encoded polypeptide; nucleotide sequence comprising: lambdoidreplication and packaging elements, said first recombination sequence,and said second recombination sequence, wherein said first and secondrecombination sequences flank a second inducible cistron for expressionof a second selectable marker transferring the polynucleotide sequenceslocated between said first and second recombination sequences of saiddonor plasmid to said recipient phage to obtain cointegrates; d)propagating said cointegrates in liquid medium selective for said firstselectable marker; and e) harvesting phages displaying said fusionpolypeptide.
 2. A process of obtaining recombinant lambdoidbacteriophage with high density display of functional peptides andproteins on surface of said bacteriophage comprising the steps of: a)Constructing a donor plasmid comprising: A first recombination sequence,and a second recombination sequence, wherein said first and secondrecombination sequences are incompatible with each other, and whereinsaid first and second recombination sequences flank a nucleotidesequence that defines the elements for replication of the plasmid inbacteria, a nucleotide sequence encoding a first selectable marker, anda first inducible cistron comprising a lambdoid capsid/exogenous proteinfusion protein encoding sequence) constructing a recipient phage havinga nucleotide sequence comprising: lambdoid replication and packagingelements, said first recombination sequence, and said secondrecombination sequence, wherein said first and second recombinationsequences flank a second inducible cistron for expression of a secondselectable marker; c) transferring the polynucleotide sequences locatedbetween said first and second recombination sequences of said donorplasmid to said recipient phage to obtain cointegrates; d) propagatingsaid cointegrates in liquid medium selective for said first selectablemarker; and e) harvesting phages displaying said fusion protein, whereinsaid plasmid has the same genetic map as shown in FIG. 2 for pVCDcDL1.3. A process of obtaining recombinant lambdoid bacteriophage with highdensity display of functional peptides and proteins on surface of saidbacteriophage comprising the steps of: a) Constructing a donor plasmidcomprising: A first recombination sequence, and a second recombinationsequence, wherein said first and second recombination sequences areincompatible with each other, and wherein said first and secondrecombination sequences flank a nucleotide sequence that defines theelements for replication of the plasmid in bacteria, a nucleotidesequence encoding a first selectable marker, and a first induciblecistron comprising a lambdoid capsid/heterologous polypeptide fusionprotein encoding sequence; b) constructing a recipient phage having anucleotide sequence comprising: lambdoid replication and packagingelements, said first recombination sequence, and said secondrecombination sequence, wherein said first and second recombinationsequences flank a second inducible cistron for expression of a secondselectable marker; c) transferring the polynucleotide sequences locatedbetween said first and second recombination sequences of said donorplasmid to said recipient phage to obtain cointegrates; d) propagatingsaid cointegrates in liquid medium selective for said first selectablemarker; and e) harvesting phages displaying said fusion protein, whereinsaid cointegrate containing recombinant phage particles comprise amatrix of proteins encapsulating a lambdoid genome encoding said fusionprotein, wherein said fusion protein is surface accessible in saidmatrix, and wherein said fusion protein comprises gpD lambdoidbacteriophage capsid polypeptide, a linker polypeptide, a proteasecleavage site, a linker poly peptide, and said heterologous polypeptide.4. A process as claimed in claim 1 wherein said heterologous polypeptideof the cointegrate-containing recombinant phage particles is selectedfrom the group consisting of capsid protein p24 of HumanImmunodeficiency Virus, decapeptide cmyc, and a single chain antibodyfragment (scFv).
 5. A process as claimed in claim 2 wherein saidheterologous polypeptide of the cointegrate-containing recombinant phageparticles is capsid protein p24 of Human Immunodeficiency Virus.
 6. Aprocess as claimed in claim 2 wherein said heterologous polypeptide ofthe cointegrate-containing recombinant phage particles is decapeptidecmyc.
 7. A process of obtaining recombinant lambdoid bacteriophage withhigh density display of functional peptides and proteins on surface ofsaid bacteriophage comprising the steps of: a) Constructing a donorplasmid comprising: A first recombination sequence, and a secondrecombination sequence, wherein said first and second recombinationsequences are incompatible with each other, and wherein said first andsecond recombination sequences flank a nucleotide sequence that definesthe elements for replication of the vector in bacteria, a nucleotidesequence encoding a first selectable marker, and a first induciblecistron comprising a lambdoid capsid/heterologous polypeptide fusionprotein encoding sequence; b) constructing a recipient phage having anucleotide sequence comprising: a lambdoid replication and packagingelements, said first recombination sequence, and said secondrecombination sequence, wherein said first and second recombinationsequences flank a second inducible cistron for expression of a secondselectable marker c) transferring the polynucleotide sequences locatedbetween said first and second recombination sequences of said donorplasmid to said recipient phage to obtain cointegrates; d) propagatingsaid cointegrates in liquid medium selective for said first selectablemarker; and e) harvesting phages displaying said fusion protein, whereinthe number of fusion protein molecules on the surface of eachrecombinant lambdoid phage particle is 100-1000 fold more than capsidprotein molecules on surface of each particle of filamentous phage M13.